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| Targets |
Biotin-VAD-FMK targets caspases, a family of cysteine-aspartic proteases that are key mediators of apoptosis. Caspases are synthesized as inactive zymogens (procaspases) and are activated by proteolytic cleavage in response to apoptotic stimuli. Once activated, caspases cleave specific substrates, leading to the characteristic morphological and biochemical features of apoptosis. Biotin-VAD-FMK contains the VAD (Val-Ala-Asp) sequence, which is recognized by caspases, and the fluoromethyl ketone (FMK) group, which forms an irreversible covalent bond with the catalytic cysteine residue of active caspases. The biotin moiety enables detection and purification of labeled caspases. The compound is a pan-caspase inhibitor, targeting multiple caspase family members including caspase-1, -3, -4, -7, and -8.
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| ln Vitro |
Biotin-VAD-FMK is a synthetic peptide formulated as a methyl ester to increase cell permeability.
In vitro, Biotin-VAD-FMK is used to identify active caspases in cell lysates and intact cells. The compound irreversibly binds to active caspases through the FMK group, allowing the detection of caspase activity by streptavidin-based methods such as Western blotting, ELISA, or flow cytometry. The compound's cell permeability (due to the methyl ester modification) enables labeling of caspases in live cells, allowing the study of caspase activation dynamics. Biotin-VAD-FMK is also used to affinity-purify active caspases and their interacting proteins for proteomic analysis. The compound is highly specific for caspases and does not label other cysteine proteases. Biotin-VAD-FMK is a standard reagent in apoptosis research. |
| ln Vivo |
Using the biotinylated caspase inhibitor biotin-VAD-fmk [2], which only detects active caspases, an in vivo affinity labeling technique was employed to investigate whether caspase-2 is activated in the absence of proteolytic cleavage.
In vivo data for Biotin-VAD-FMK are limited as the compound is primarily used in in vitro and ex vivo applications. The compound's cell permeability and irreversible binding make it potentially useful for in vivo labeling of active caspases, but its use is typically limited to cell culture and tissue lysate applications. The compound is intended for research use only and is not for human therapeutic applications. It is not administered to live animals in most research settings. The pharmacokinetic properties, bioavailability, and tissue distribution have not been characterized. |
| Enzyme Assay |
In vitro enzyme assays for Biotin-VAD-FMK involve assessing the inhibition and labeling of caspase activity. Recombinant caspases or cell lysates containing active caspases are incubated with Biotin-VAD-FMK at concentrations of 0.1-10 microM for 1-2 hours at 37degC. The compound irreversibly binds to the catalytic cysteine of active caspases. The labeled caspases are then detected by Western blotting using streptavidin-HRP (horseradish peroxidase) or by ELISA using streptavidin-coated plates. For activity assays, the inhibition of caspase activity is measured using fluorogenic substrates such as Ac-DEVD-AMC (for caspase-3/7) or Ac-YVAD-AMC (for caspase-1). The degree of inhibition and labeling is determined. The specificity of labeling is confirmed by competition with non-biotinylated VAD-FMK.
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| Cell Assay |
In vitro cellular assays for Biotin-VAD-FMK are performed using cells undergoing apoptosis or with constitutively active caspases. Cells (e.g., Jurkat, HeLa, or primary cells) are cultured in appropriate medium and treated with apoptotic stimuli (e.g., staurosporine, etoposide, TNF-alpha/cycloheximide) to induce caspase activation. Cells are then incubated with Biotin-VAD-FMK (1-10 microM) for 30-60 minutes at 37degC. After washing, cells are fixed and permeabilized for flow cytometry analysis using streptavidin conjugated to a fluorophore (e.g., FITC, PE, APC). Alternatively, cell lysates are prepared and analyzed by Western blotting with streptavidin-HRP. The biotin-labeled caspase bands are detected, and the pattern of caspase activation is characterized. The compound allows the identification of which caspases are activated in response to specific stimuli.
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| Animal Protocol |
In vivo animal studies for Biotin-VAD-FMK are not typically performed as the compound is used as a research reagent for in vitro and ex vivo applications. The compound is not intended for administration to live animals. For in vivo studies of caspase activity, researchers typically use non-biotinylated caspase inhibitors or activity-based probes that are optimized for in vivo use. Biotin-VAD-FMK is used to study caspase activity in tissue lysates and cell extracts. Researchers interested in in vivo applications should consult the primary literature for the most current information.
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| ADME/Pharmacokinetics |
Biotin-VAD-FMK (CAS#: 1135688-15-1) has molecular formula C30H4₉FN₆O₈S and molecular weight 672.81. The compound is a cell-permeable, irreversible, biotin-labeled caspase inhibitor. It contains the VAD (Val-Ala-Asp) sequence for caspase recognition, the FMK (fluoromethyl ketone) group for irreversible covalent binding to the catalytic cysteine, and a biotin moiety for detection and affinity purification. The compound is designed as a methyl ester to facilitate cell permeability. It is used to identify and characterize active caspases in cell lysates and intact cells. Biotin-VAD-FMK is supplied as a white to light yellow solid with ≥95% purity. Storage: -20degC for up to 3 years. The compound is intended for research use only.
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| Toxicity/Toxicokinetics |
Toxicological information for Biotin-VAD-FMK is limited as the compound is a research reagent. As a caspase inhibitor, the compound may interfere with apoptosis and other caspase-dependent processes. The compound should be handled with standard laboratory precautions including the use of personal protective equipment and working in a well-ventilated area. Avoid inhalation, ingestion, and skin contact. The compound should be stored at -20degC and disposed of according to institutional guidelines for chemical and biological waste.
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| References |
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| Additional Infomation |
Biotin-VAD-FMK (CAS#: 1135688-15-1) is a cell-permeable, irreversible, biotin-labeled pan-caspase inhibitor used as a probe for detecting and characterizing active caspases. The compound contains the VAD sequence for caspase recognition, the FMK group for covalent binding to the catalytic cysteine, and a biotin moiety for streptavidin-based detection and affinity purification. Biotin-VAD-FMK is widely used in apoptosis research to study caspase activation, identify active caspase species, and purify caspase-containing complexes for proteomic analysis. The compound is supplied for research use only and is not approved for clinical or therapeutic applications. As of the current date, Biotin-VAD-FMK remains a research reagent.
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| Molecular Formula |
C30H49FN6O8S
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| Molecular Weight |
672.8089
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| Exact Mass |
672.331
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| CAS # |
1135688-15-1
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| PubChem CID |
25108682
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| Appearance |
White to light yellow solid powder
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| LogP |
0.8
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
22
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| Heavy Atom Count |
46
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| Complexity |
1100
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| Defined Atom Stereocenter Count |
6
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| SMILES |
S1C([H])([H])[C@@]2([H])[C@@]([H])([C@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])[C@]([H])(C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]([H])(C(C([H])([H])F)=O)C([H])([H])C(=O)OC([H])([H])[H])=O)=O)C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)N([H])C(N2[H])=O
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| InChi Key |
MOQLQMKJGYKHCT-VRKQFSAKSA-N
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| InChi Code |
InChI=1S/C30H49FN6O8S/c1-17(2)26(29(43)33-18(3)28(42)34-19(21(38)15-31)14-25(41)45-4)36-24(40)12-6-5-9-13-32-23(39)11-8-7-10-22-27-20(16-46-22)35-30(44)37-27/h17-20,22,26-27H,5-16H2,1-4H3,(H,32,39)(H,33,43)(H,34,42)(H,36,40)(H2,35,37,44)/t18-,19-,20-,22-,26-,27-/m0/s1
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| Chemical Name |
methyl (3S)-3-[[(2S)-2-[[(2S)-2-[6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]-5-fluoro-4-oxopentanoate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 27.3 mg/mL (~40.58 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4863 mL | 7.4315 mL | 14.8630 mL | |
| 5 mM | 0.2973 mL | 1.4863 mL | 2.9726 mL | |
| 10 mM | 0.1486 mL | 0.7432 mL | 1.4863 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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